We will continue our studies on gene expression using the cloned Chinese hamster gene for thymidine kinase, which we have recently isolated as a DNA probe. Restriction enzyme analysis will be used to determine DNA methylation patterns in or near the TK locus for cells with different levels of expression of thymidine kinase. Two problems to be addressed will be: (1)\whether the timegraded acquisition of BUDR resistance which we have described previously in Chinese hamster cells results from gradual methylation of strategic DNA sites, shutting down expression of thymidine kinase, and (2)\whether reexpression of TK in BUDR-resistant cells after exposure to such diverse agents as 5-azacytidine, butyrate, and hypertonic glycine or NaCl is the common result of hypomethylation at these sites. During the past year we have completed our study of proline independence in CHO-K1 Chinese hamster cells, long regarded as a prototype for auxotrophic mutations in mammalian cells. We have shown that treatment by 5-azacytidine induces proline-independent variants in high frequency with coordinate expression of pyrroline-5-carboxylase and ornithine aminotransferase activities. In this system, as in others we have previously studied (thymidine kinase, asparagine synthetase), variation appears to be based on stable changes in gene expression which masquerade as genetic mutations. Although little known up to the present time, such epigenetic variations may play an important role along with genetic changes in transformation and carcinogenesis. (P)